JP2001502910A - Enzymatic coenzyme cycling using soluble pyridine nucleotide transhydrogenase - Google Patents

Abstract

The present invention pertains to an enzymatic reaction involving a pyridine nucleotide cofactor, wherein an enzyme is used that has a sequence of greater than 70% identity to SEQ ID NO: 2 and is capable of transferring reducing equivalents between pyridine nucleotide cofactors. Alternatively, a cell transformed to express the enzyme may be used.

Description

BACKGROUND OF THE INVENTION Field of the Invention The enzymatic coenzyme cycling invention using soluble pyridine nucleotide transhydrogenase, in vivo or in vitro enzymatic reactions, in the analysis technique, for example by enzymatic or whole cell biotransformation or enzyme, pyridine nucleotides Related to the use of enzymes for the oxidation or reduction of coenzymes. BACKGROUND OF THE INVENTION Biotransformation procedures using natural or genetically modified microbial organisms or isolated enzymes provide a method for the synthesis of many useful substances. Bioconversion has several advantages over chemical synthesis methods, particularly the site and stereospecificity of enzyme catalysis, the use of mild reaction conditions, and the elimination of harmful solvents. Oxidoreductase enzymes often require a redox-active coenzyme for activity. The most common of such coenzymes are the pyridine nucleotide coenzyme nicotinamide adenine dinucleotide (NAD: oxidized NAD ⁺ , reduced NADH) and nicotinamide adenine dinucleotide phosphate (NAD P: oxidized NADP ⁺ , Reduced NADPH). These coenzymes are expensive and cannot be practically supplied in stoichiometric quantities unless they are particularly valuable products. This is one reason that many oxidoreductases are used exclusively in bioconversion reactions. Providing a means for regenerating the desired form of coenzyme can reduce the need for coenzyme in the bioconversion process. This means that the coenzyme needs to be supplied in catalytic amount only. For example, if the reaction of interest requires NAD ⁺ , which is reduced to NADH in that reaction, this NADH may be converted by another enzyme system, such as NAD ⁺ -dependent formate dehydrogenase in the presence of formate. NAD ⁺ can be oxidized again. This is called coenzyme cycling. Formate dehydrogenase is particularly suitable for this application because its catalytic reaction is essentially irreversible. To complicate matters further, most of the NAD-requiring enzymes cannot use NADP as a coenzyme, and vice versa. For example, formate dehydrogenase cannot be used to regenerate NADPH from NADP ⁺ . In a special case, the bioconversion process requires two oxidoreductases that require separate coenzymes. For example, the recently proposed bioconversion process of converting morphine to the potent analgesic hydromorphone requires the continuous action of NADP ⁺ -dependent morphine dehydrogenase and NADH-dependent morphinone reductase (French et al (1995) Bio / Technology 13: 674-676). In the first reaction, morphine is converted to morphinone with reduction of NADP ⁺ to NADPH, and then in the second reaction morphine is converted to hydromorphone with oxidation of NADH to NAD ⁺ . Therefore, both NADP ⁺ and NADH must be supplied. To complicate matters, morphine dehydrogenase reduces the hydromorphone product to the undesired product dihydromorphine, with re-oxidation of NADPH to NADP ⁺ in the presence of NADPH produced in the first reaction. These reactions are shown in the attached FIG. 1A. Pyridine nucleotide-dependent enzymes are also used in certain enzyme assays where the amount of analyte is determined by the degree of oxidation or reduction of the coenzyme. Oxidation and reduction of NAD and NADP are measured by several methods, such as spectroscopy and fluorimetry. However, for detection, particularly sensitive oxidation or reduction, only NAD or NADP is available, but not both. For example oxidation of NADH to NAD ⁺ using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerokinase (PGK) enzyme, adenosine diphosphate in the reaction that is dependent on the presence of NA D ⁺ a (ADP) Phosphorylation to adenosine triphosphate (ATP), and then the resulting ATP is detected by the ATP-dependent luminescence reaction of firefly luciferase, which can be detected with extremely high sensitivity. Since commercially available GAPDH is specific for NAD ⁺ , the oxidation of NADPH to NADP ⁺ cannot be detected using this technique. Some of the problems described above can be solved by using enzymes that transfer the reduction equivalent between NAD and NADP, for example, by oxidizing NADPH to NADP ⁺ while reducing NADf to NADH. Such an enzyme is known as pyridine nucleotide transhydrogenase (PNTH). Several classes of enzymes exhibit this activity (Rydstrom et al (1987) in "Pyridine nucleotide coenzymes: chemical, biochemical and medical aspects", part B, eds. Dolphin et al, John Wiley and Sons, NY, p. 433-460). The best known are the membrane-bound proton pumps PNTH found in the membranes of mitochondria and certain bacteria, such as E. coli. This enzyme is membrane bound and is generally not suitable for bioconversion or analytical purposes. Soluble non-energy related PNTH has been reported to be present in certain bacteria such as, for example, Pseudomanas fluorescens, Pseudomonas aerugjnosa and Azotobacter vinelandii. Although this enzyme has some known properties, its use is limited. SUMMARY OF THE INVENTION The gene encoding the soluble transhydrogenase of Pseudomonas fluorescene NCIMB 9815 (designated sth) was cloned and sequenced, and the enzyme was overexpressed in Escherichia coli. This makes it relatively easy to prepare a large amount of enzyme. This enzyme was purified and characterized. The enzyme reacts in the catalyzed reaction, ie, transfer of reducing equivalents between NAD and NADP or between analogs of these coenzymes; the nucleotide sequence of the structural gene sth encoding this enzyme, and the derivation from that sequence. the deduced amino acid sequence of octopus enzyme; containing approximately 50,000 subunits M _r of the structural properties of the enzyme; ability to form a giant polymer M _r exceeding and 1,000,000, it is defined by. A first aspect of the present invention is to use this enzyme to act on pyridine nucleotide coenzymes to accelerate the bioconversion process, for example, by altering the oxidation state of NAD or NADP or analogs of these coenzymes. is there. In this way, another enzyme can act on these coenzymes. Alternatively, an altered form of the enzyme prepared by random or site-directed mutagenesis of the structural gene may be used. Enzymes so altered may exhibit altered levels of activity, altered regulation, altered subunit structure. This gene sth constitutes the second aspect of the present invention. The gene can be used to produce the enzyme and variants of the enzyme using genetically modified organisms. For example, genetically modified organisms having the sth gene as all or part of a heterologous construct can be grown in such a way as to promote production of the enzyme, after which the enzyme is recovered from the medium or cell extract. be able to. Methods for accomplishing this are well-known in the art. A third aspect of the present invention is a genetically modified organism that expresses this enzyme. Such organisms can be used in whole cell bioconversion processes and can facilitate this process by the presence of active enzymes in the cells. Techniques for producing such recombinant organisms are well known in the art. According to a fourth aspect of the invention, the enzyme is used in an enzyme-based analytical assay to enhance these assays. For example, the enzyme, in fact, may use the signal to be measured in the case of oxidation of NAD PH to NADP ^+, to convert NADH to a signal that can be measured based on the oxidation of NA D ^+. This converted signal can then be detected with more sensitive techniques not previously applicable. DESCRIPTION OF THE INVENTION The present invention can be used with enzymes having the sequence shown in SEQ ID NO: 2, or an amino acid sequence having 70% or more, preferably at least 80%, more preferably at least 90% identity. This enzyme can be used by itself or as a kind of transformed organism. Suitable hosts for transformation are well known to those skilled in the art. An example of a suitable host is E. coli. The enzymes or organisms of the present invention are used in bioconversion for analytical or other suitable purposes. The invention is particularly useful when the enzyme is involved in a reaction using a pyridine nucleotide coenzyme. A specific example is shown in FIG. 1B (compare FIG. 1A). The use of STH indicates that avoiding the construction of NADH greatly reduces the reduction of hydromorphine. This eliminates the need to supply expensive coenzymes. In bioconversion, STH reciprocates transports reducing equivalents from NADH to NAD ⁺ , thus allowing the cells to be used more than once in this process. Example 1 below illustrates the cloning and sequencing of sth, while Examples 2 and 3 illustrate the use of STH of the present invention. These examples refer to FIG. 1 (above) and other accompanying figures below: FIG. 2 shows a 5.0 kb Eco R1 fragment and a 1.5 kb SacII / XhoI subclone with the sth gene. It is a restriction map. Filled areas are coding regions, and arrows represent sequencing reactions. FIG. 3 shows the conversion of morphine to hydromorphone in the presence of soluble transhydrogenase. Squares are morphine, circles are hydromorphinone, triangles are dihydromorphine. FIG. 4 shows the sequential morphine bioconversion by E. coli JM109 / pMORB3-AmutMC80S / pPNT4 and E. coli JM109 / pMORB3-AmutMC80S cells (OC = opiate concentration (mM), □ = morphine, ● = hydromorphone, ▲ = Dihydromorphine). Example 1 Thionicotinamide adenine dinucleotide (tNAD ⁺ ) and adenosine-2 ′, 5′-diphosphate agarose were obtained from Sigma (Poole, Dorset, UK). Other reagents were of analytical or high purity and were obtained from Sigma or Aldrich (Gillingham, Dorset, UK). Pseudomonas fluorescens NCIMB9815 was obtained from the National Collection of Industrial and Marine Bacteria (Aberdeen, Scotland, UK). Escherichia coli JM109 was obtained from Promega (Southampton, UK). At 30 ° C. with both the biological shaken rotated at 180rpm (P.fluorescens) or 37 ℃ (E.coli), SOB medium (Sambrook et al (1989) Molecular Cloning:. A Laboratory Manual, 2 nd edn, Cold (Spring Harbor Laboratory Press, Cold Spring Harbor, NY). STH activity was measured in a reaction mixture consisting of 0.1 mM tNA D ⁺ and 0.1 mM NADPH at 30 ° C. in 50 mM phosphate buffer, pH 7.0, at 30 ° C., using thionicotinamide adenine dinucleotide (tNAD ⁺ ), an NAD ⁺ analog. Reduction was observed by 400 nm spectral property change and quantified in the usual manner. One unit (U) of the enzyme activity is defined as the amount of activity that reduces 1 mmol of tNAD ⁺ per minute under the above conditions. The molar change of tNAD ⁺ upon reduction to tNADH was 11 3001 mol ⁻¹ cm ⁻¹ at an absorbance of 400 nm (Cohen et al (1970) J. Biol. Chem. 245: 2825-2836). Protein concentration was quantified by Pierce reagent (Rockford, II, USA) according to the manufacturer's protocol in the usual manner. Bovine serum albumin was used as a reference. Specific activity was calculated in units of STH activity per mg of protein (U / mg). Obtained a standard cloning vector, p Bluescript SK +, from Stratagene (Cam bridge, Cambs., UK). PS IEMBL, a low copy number vector, is available from Poustka et al (1984) Proc. Nalt. Acad. Sci. USA. 81: 4129-4133. Southern blotting and DNA manipulations were performed using standard techniques (Sambrook et al, supra). Purification of STH: Soluble pyridine nucleotide transhydrogenase (STH) was obtained from Hojeberg et al (1976) Eur. J. Biochem. 66: 467-475, and purified from cells of P.fluorescens NCIMB9815 by a modified method. Cells were grown to stationary phase in 11 of SOB medium. The cells were collected by centrifugation (5000 g, 15 min) and resuspended in 20 ml of buffer A (50 mM Tris / HCl, pH 7.0, supplemented with 2 mM dithiothreitol). The cells were then triturated using MS E Soniprep 150 by sonication (open for 30 seconds to cool in an ice bath and burst 12 times 5 seconds for 5 seconds). Cell debris was removed by centrifugation (25,000 g, 10 minutes). The extract contained 93 units of STH activity at a specific activity of 0.19 U / mg. STH was purified using a 1 cm id column (7.6 cm height) packed with 6 ml of adenosine-2 ', 5' .- diphosphate agarose. The column was controlled and operated so as to be 12 ml / h during use and 24 ml / h during washing. All manipulations were performed at 4 ° C., and all buffers contained 2 mM dithiothreitol. After equilibrating the column with 5 mM CaCl ₂ in buffer A, the crude extract (20 ml) adjusted to a final concentration of 5 mM by adding CaCl ₂ was passed through the column. Then buffer A 0.4 M NaCl column, 5 mM CaCl ₂ 90 ml, then buffer A 0.7M NaCl, washed with 5 mM CaCl ₂ 24 ml. Elution in the reverse direction was also with 50 mM Tris / HCl, pH 8.9 containing 0.4 M NaCl. Each 5 ml fraction was collected and the active fraction was collected. The collection was prepared using the Amicon 8050 ultrafiltration cell fitted membrane nominal M _r cutoff of 10,000 and concentrated ultrafiltration, then diafiltered using buffer A, pH and The salt concentration was reduced. Final volume was 1.5 ml. The product contained 62 U of STH activity at a specific activity of 140 U / mg. The product was then applied to a gel filtration column 1.6 cm id and 75 cm high packed with 150 ml Sephacryl S-300 (Pharmacia) equilibrated with buffer A. The column was controlled and operated to 8 ml / h. Two ml fractions were collected. Active fractions (16 ml) were collected, ultrafiltered and concentrated to a final volume of 1 ml as described above. This product contained 26 U of STH activity at a specific activity of 310 U / mg. Prior to analysis by SDS-PAGE, the samples were lyophilized, further concentrated and resuspended in a small amount of buffer A. This reconstituted feed had no activity. SD S-PAGE showed Pseudomonas aeruginosa (Rydstrom et al, supra ) single protein band at M _r 55,000 apparent that matches the reported value for the origin of the enzyme. Cloning: Proteins were poly (vinylidene difluoride) (PVDF) membranes from SDS-PAGE gels using the Phast Transfer semi-dry transfer system (Pharmacia, St. Albans, Herts., UK) according to the manufacturer's instructions. (Problott, Applied Biosystems, Foster City, CA, USA). N-terminal sequence was determined from automated Edman degradation. The N-terminal sequence of the purified PNTH was determined as follows: AVYNYDVVVLGS- (G / V) -PAGE- (G / V) -AAMNAA- (R / D)-The parentheses indicate that the assignment is uncertain. Show. A P. fluorescens codon bias table was obtained based on 20 genes in the Gen-ENBL database. This revealed that G and C at position 3 were significantly preferred for most codons. Based on this codon bias, the following degenerate oligonucleotides were designed: AC- (C / G) AC- (C / G) AC-GTC-GTA-GTT-GTA- (C / G) AC- (G / C) GC (based on residues 1-9 of the N-terminal sequence). A genomic DNA Southern blot of P. fluorescens NCIMB9815 showed that this oligonucleotide bound most strongly to the 5.0 kb Eco R1 fragment. A 4-6 kb Eco R1 fragment library was prepared in the cloning vector pBluescript using E. coli JM109 as a host and recombinant cells were screened by colony blotting using oligonucleotide probes. Isolation of some positive colonies showed that they all had the same 5.0 kb insert. Both orientations of the insert were recovered. These recombinant plasmids were named pSTH1A and pSTH1B with only the orientation of the Eco R1 insert changed. The location of the sth gene was determined by restriction mapping of the insert followed by Southern analysis using oligonucleotide probes. Sequencing showed the presence of an open reading frame encoding the same N-terminal sequence of the protein as determined by STH. Various subclones were made with pBluescript SK + and sequenced using vector-based primers, as shown in FIG. The sequence of sth and the deduced amino acid sequence of sth are shown in SEQ ID NOs: 1 and 2. E. Cell extracts obtained from saturated cultures of E. coli JM109 / pSTH1A or pSTH1B showed detectable STH activity when assayed by reduction of thionicotinamide adenine dinucleotide (tNAD ⁺ ) in the presence of NA DPH. The 1.5 kb SacII / XhoI fragment of pSTH1A was subcloned with pBluescript SK + (FIG. 2). This plasmid was named pSTH2. In pSTH2, sth was in the correct orientation expressed from the lac promoter of pBluescript SK +. In the absence, presence, or presence of 0.4 mM IPTG, E. coli. Cell extracts obtained from saturated cultures of E. coli JM109 / pSTH2 exhibited transhydrogenase activities of 4.1 U / mg and 22.0 U / mg, respectively. Based on the specific activity of purified STH, in the latter case, STH was assessed to have formed about 6% of soluble cellular proteins, corresponding to about 100 cultures of the level observed in P. fluorescens. Recombinant STH was purified using adenosine-2 ', 5'-diphosphate agarose in a single affinity chromatography step until it was apparently homogeneous. Cell extracts were prepared as described above from 1 liter of a saturated culture of E. coli JM109 / pSTH2 grown in the presence of 0.4 mM IPTG. Of 25 ml of the obtained cell extract, 5 ml containing 27 U / mg of specific activity and 2140 U of STH activity was applied to a column packed with adenosine-2 ', 5'-diphosphate agarose as described above. The column, buffer A 0.7M NaCl, washed with 5 mM CaCl ₂ in 35 ml. The STH was then eluted with 0.4 M NaCl in 50 mM Tris / HCl pH 8.9. The most active fractions, a total volume of 13 ml, were collected and concentrated and diafiltered as described above except that a membrane with a nominal molecular weight cutoff of 30,000 was used. The product has 900 U STH activity at a specific activity of 300 U / mg. This material appeared homogeneous on SDS-PAGE; therefore, the gel filtration step was omitted. The purified STH was stored at −20 ° C. in buffer A containing 2 mM dithiothreitol, but after several weeks no decrease in activity was detected. The properties of the recombinant STH were compared to those reported for the enzyme from Pseudomon asaeruginosa. The subunit Mr measured by SDS-PAGE is consistent with previous reports (Rydstrom et al, supra). To determine if the recombinant enzyme is capable of forming a large polymer, the sample was adsorbed on a carbon film, negatively stained with 1% w / v uranyl acetate, and electron-scanned using a Phillips CM100 electron microscope. Examined microscopically. Long chain polymers with a diameter of about 10 nm and a length of more than 500 nm have been discovered. This is consistent with the previous report (Louie et al (1972) J. Mol. Biol. 70: 651-664). Example 2 Morphine dehydrogenase and morphinone reductase were prepared from recombination of recombinant strains of Escherichia coli. (See Willey et al (1993) Biochem. J. 290: 539-544; French and Bruce (1995) Biochem. J. 312: 671-678). STH was prepared from Pseudomonas fuorescens NCIMB9815 by the method described in Example 1. Morphine alkaloids were quantified by HPLC (French et al, supra). Reaction mixture obtained by adding 10 mM morphine, 0.2 mM NADPH, 0.2 mM NAD ⁺ , 1 mM dithiothreitol, 1 unit of morphinone reductase, 1 unit of morphine dehydrogenase, and 6 units of STH to 0.5 ml of 50 mM Tris / HCl buffer. Was kept at 4 ° C. for 8 hours. 50 μl samples were taken at regular intervals, treated with acetic acid to precipitate proteins and analyzed by HPLC. As shown in FIG. 3, morphine was converted to hydromorphone in high yield. In parallel, experiments with STH deficiency were also performed. In this case, no conversion of morphine has occurred. This behavior indicates that STH is capable of catalyzing the coenzyme cycle during the enzymatic bioconversion process. Example 3 A 1.2 kb PstI fragment carrying the mutant morphine dehydrogenase structural gene (morA) was complemented with an upstream ribosome binding site, and the promoter sequence was reduced to a single copy vector, pSI, previously digested with PstI. When ligated with IEMBL to create the pMO RA4mutMC80S construct, it contained appropriate restriction sites for further subcloning. A mutated morA gene, a 1.2 kb HindIII / Eco R1 fragment having a ribosome binding site and a promoter site was excised from pMORA4mutMC80S, and a single copy of morB and a structural gene of morphinone reductase were combined with its ribosome binding site and promoter region. The HindIII / Eco R1-digested and ligated into pMORB3 (French et al, supra) to create the pMORB3-AmutMC80S construct. A 1.5 kb PstI / XhoI fragment carrying the structural gene for soluble pyridine nucleotide transhydrogenase was previously digested with PstI and SalI. It was ligated to pS1EMB L to generate pRNT4. E. coli JM109 / pMORB3-AmutMC80S and E. coli JM109 / pMORB3-AmutMC80S / pPN T4 cells were grown to stationary phase and collected by centrifugation at 17,310 g for 15 minutes at 4 ° C. The cells were then washed with 50 mM Tris HCl (pH 7.5) and centrifuged again. The supernatant was removed and the pelleted cells were stored on ice until needed for bioconversion. Typical values of enzyme activity in E. coli JM109 / pMORB3-AmutMC80S cells are 0.06 U / mg for morphine dehydrogenase and 0.88 U / mg for morphinone reductase, while E. coli In JM109 / pMORB3-AmutMC80 / pPNT4 cells, the morphine dehydrogenase concentration was 0.044 U / mg, morphinone reductase was 0.78 U / mg, and STH was 0.72 U / mg. Small-scale whole-cell bioconversion (3 ml total) was performed in a reaction mixture containing 20 mM morphine in 50 mM Tris HCl (pH 7.5) and a final cell density of 0.17 g / ml. Bioconversion was performed in duplicate on a rotary shaker at 30 ° C. and samples were taken at regular intervals. Samples were clarified by centrifugation and opiate content was analyzed using HPLC as described previously (French et al, supra). A series of sequential bioconversions was performed using the same batch of cells collected and washed during the incubation. The results shown in FIG. 4 indicate that cells containing recombinant STH are capable of being used more than once in the bioconversion process, whereas cells lacking recombinant STH can be used only once. These results indicate that recombinant STH is capable of behaving as a coenzyme cycling in an ex vivo enzyme process dependent on NADP and NAD.

[Procedure of Amendment] Article 184-8, Paragraph 1 of the Patent Act [Submission date] November 6, 1998 (1998. 11.6) [Correction contents]                                The scope of the claims 1. A sequence having 70% or more identity to SEQ ID NO: 2 and Transformed to express an enzyme capable of transferring reducing equivalents between leotide coenzymes An unnatural organism that has been replaced. 2. The enzyme is a soluble pyridine nucleotide transhydrogenase Item 2. An organism according to item 1. 3. Use of the organism according to claim 1 or 2 as a biocatalyst. 4. A soluble pyridine nucleic acid having at least 70% identity to SEQ ID NO: 1 Nucleotides encoding an enzyme having otide transhydrogenase activity Child. 5. An enzyme or an organism comprising the use of an enzyme or organism as defined in claim 1 or 2. And a method for converting a substrate into a product by a pyridine nucleotide coenzyme. 6. 6. The method of claim 5, which is a bioconversion or an assay. 7. The method according to claim 5 or 6, wherein the substrate is morphine. 8. 8. The method according to claim 5, wherein the coenzyme is used in a catalytic amount.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/02 C12P 17/04 // C12P 17/04 C12N 5/00 A (81) Designated country EP (AT , BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA , GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AU, AZ, BA, BB, BG, BR, BY, CA, CN, CU, CZ, EE, GB, GE, GH, HU, ID, I , IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, RO, RU, S D, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor French, Christopher Edward UK, CB 2.1 Cutty, Ke Bridge, Tennis Court Road, Institute of Biotechnology, University of Cambridge

Claims (1)

[Claims] 1. A sequence having 70% or more identity to SEQ ID NO: 2 and Transformed to express an enzyme capable of transferring reducing equivalents between leotide coenzymes The replaced organism. 2. The enzyme is a soluble pyridine nucleotide transhydrogenase Item 2. An organism according to item 1. 3. Use of the organism according to claim 1 or 2 as a biocatalyst. 4. A soluble pyridine nucleic acid having at least 70% identity to SEQ ID NO: 1 Nucleotides encoding an enzyme having otide transhydrogenase activity Child. 5. An enzyme or an organism comprising the use of an enzyme or organism as defined in claim 1 or 2. And a method for converting a substrate into a product by a pyridine nucleotide coenzyme. 6. 6. The method of claim 5, which is a bioconversion or an assay. 7. The method according to claim 5 or 6, wherein the substrate is morphine. 8. 8. The method according to claim 5, wherein the coenzyme is used in a catalytic amount.